ABSTRACT Acute respiratory distress syndrome (ARDS) is a serious lung condition characterized by airspace flooding and widespread inflammation. Survival of ARDS is chiefly attributed to the ability to maintain airspace fluid balance. This involves two complimentary processes: sodium-driven fluid clearance and regulation of fluid diffusion through the epithelial barrier. The severity and risk of ARDS is magnified with chronic alcohol abuse. In the alcoholic lung, paracellular diffusion is increased, resulting in fluid leakage into the lung, and thus compensatory fluid clearance is necessary to maintain fluid balance, leaving the alcoholic lung primed for ARDS. Currently, our lab is investigating the molecular mechanisms behind increased incidence of ARDS, with particular interest in the effects on tight junctions. Tight junctions serve as the major functional unit of the epithelial barrier and are crucial in providing the selective permeability by which both fluid clearance and diffusion are achieved. Previous work in the Koval lab determined that chronic alcohol ingestion increases expression of tight junction protein claudin-5 by the alveolar epithelium, which is necessary and sufficient to decrease alveolar epithelial barrier function. This impairment of the alveolar epithelial barrier correlated to molecular rearrangement of claudin-18 into spike-like structures perpendicular to the cell junction interface. These ?tight junction spikes? (TJ spikes) appear to be active areas of junction remodeling driven by increased endocytosis of tight junction proteins. Treatment with the endocytosis inhibitor Dynasore, which targets the actin-binding protein dynamin, significantly reduces the number of TJ spikes. This suggests a role for clathrin- mediated, dynamin-dependent endocytosis in TJ spike formation. However, Dynasore is still capable of inhibiting clathrin-mediated endocytosis in dynamin triple-knockout mouse fibroblasts, suggestive of Dynasore's off-target effects. Defining roles of one or more dynamin isoforms in TJ spike formation requires a more detailed molecular analysis. We hypothesize that dynamin induces the rearrangement of claudin-18 into TJ spikes that increase paracellular leak. We plan to test our hypothesis through the following aims. In Aim 1, we will examine the requirement of dynamin in TJ spike formation by manipulating dynamin expression in alveolar epithelial cells. It is also possible that TJ spike formation is due to other proteins beyond dynamin. In order to elucidate the molecular mechanism behind TJ spike formation, more candidates in addition to dynamin must be explored. In Aim 2, we will identify proteins that interact with claudin-18 that are associated with promoting TJ spike formation using complementary techniques to assess TJ protein interactions in situ. The long-term goal is to identify novel therapeutic targets to improve barrier function by redirecting spike- associated claudin-18 into barrier forming tight junctions.